CN111050774A

CN111050774A - Method for removing undifferentiated pluripotent stem cells

Info

Publication number: CN111050774A
Application number: CN201880038229.0A
Authority: CN
Inventors: 吕仁; 林禹岑
Original assignee: Academia Sinica
Current assignee: Academia Sinica
Priority date: 2017-06-07
Filing date: 2018-06-06
Publication date: 2020-04-21
Also published as: TW201920656A; WO2018226797A1; EP3634425A4; EP3634425A1; US20210147796A1; TWI719315B

Abstract

The present invention relates to a method for removing undifferentiated embryonic stem cells. In particular, the present invention relates to a method for removing undifferentiated embryonic stem cells using Cardiac Glycosides (CGs). The invention also relates to a method for preparing differentiated cells, wherein undifferentiated embryonic stem cells are removed, and to a method for cell therapy using such differentiated cells.

Description

Method for removing undifferentiated pluripotent stem cells

RELATED APPLICATIONS

The present application is entitled to U.S. provisional patent application No. 62/516,437, filed on 2017, 6/7, pursuant to 35 u.s.c. § 119, the disclosure of which is incorporated herein by reference.

Technical Field

The present invention relates to a method for removing undifferentiated pluripotent stem cells. In particular, the present invention relates to a method for removing carcinogenic undifferentiated pluripotent stem cells using Cardiac Glycosides (CGs). The invention also relates to a method for preparing differentiated cells, wherein undifferentiated pluripotent stem cells are removed, and to a method for cell therapy using such differentiated cells.

Background

Human embryonic stem cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs) are human pluripotent stem cells (hPSCs) that have unique self-renewing (almost unlimited replicative capacity) and pluripotent (capable of differentiating into all cell types of the human body except placental cells) properties. These capabilities make hPSCs promising for regenerative therapy¹. However, there are still substantial challenges to overcome before applying hPSCs to cell therapy. An important safety issue for hPSCs is their tumorigenic risk, since these cells can form teratomas upon in vivo injection at ectopic sites^2,3. Thousands of undifferentiated hPSCs present in millions of differentiated cells are sufficient to induce teratomas in a mouse model⁴. Therefore, it is important to remove all or most of the residual undifferentiated hPSCs that have potential for teratomas prior to clinical use with hPSC-derived cells.

There are several strategies to selectively remove hPSCs. These methods include the use of cytotoxic antibodies^5,6Specific antibody cell sorting^7-9Genetic manipulation^10-12And pharmacological methods^13-16. However, each method has certain disadvantages, such as high cost (cell sorting of cytotoxic antibody and specific antibody), difference between different batches (cell sorting of cytotoxic antibody and specific antibody)^17,18Non-specific binding (cytotoxic antibodies)^18-20Genetic manipulation of virulence genes and the need for stable integration (genetic manipulation), and time-consuming procedures (genetic manipulation, specific antibody cell sorting, and cytotoxic antibodies). Although many studies have attempted to prevent or prevent teratoma formation from the remaining hPSCs, clinically applicable strategies for eliminating teratoma formation remain to be developed^2,21。

In contrast, the small molecule approach has several advantages: these methods are robust, efficient, rapid, simple, and inexpensive, and do not require the insertion of genes into cells. Certain small molecules have been shown to inhibit teratoma formation in hPSCs. Stearoyl-coa desaturase inhibitor plurasin #1 prevents teratoma formation¹⁵. stearoyl-CoA desaturase, however, is a key enzyme in the biosynthesis of monosaturated fatty acids and is essential for hPSC survival¹⁵. N-benzyl nonanamide JC011 induces ER stress (stress) through PERK/AT4/DDIT3 pathway²². Chemical inhibitors of survivin, such as quercetin (quercetin) and YM155, induce selective cell death and effectively inhibit teratoma formation¹⁴. However, none of these drugs have been specifically defined or approved by the FDA.

Cardiac Glycosides (CGs) (also known as cardiac steroids, CSs) belong to a large family of compounds that can be derived from natural products. Although these compounds have different structures, they share a common structural motif. These compounds are transmembrane sodium pumps (Na)⁺/K⁺-ATPase). CGs inhibit Na⁺/K⁺ATPase, followed by an increase in the intracellular concentration of calcium ions²³. These compounds are useful as positive cardiotonic agents (positive inotropic agents), and members of this group have been used for over 200 years in the treatment of heart failure. One member of this family, digoxigenin (digoxin), is currently still in clinical use²⁴. In addition, CGs are currently believed to have potential therapeutic perspectives in cancer therapy²⁵. Several research reports indicate that CGs play an important role in inducing cell death in several cancer cells²³. Cancer cells are more susceptible than normal tissue cells. The molecular mechanism may be Na in cancer cells⁺/K⁺Overexpression of the α subunit specific for ATPase²⁶. Those studies indicate that CGs are selective for cell type and can distinguish between normal and transformed cells.

Although cardiac glycosides act as multiple signal sensors, no study has been made to investigate whether these drugs can eliminate undifferentiated PSCs while retaining their progeny or differentiated cells.

Disclosure of Invention

In the present invention, it has been unexpectedly found that Cardiac Glycosides (CGs) specifically induce cell death in human embryonic stem cells (hESCs), but do not occur in differentiated cells or hESC-derived Mesenchymal Stem Cells (MSCs). It was also found that Cardiac Glycosides (CGs) significantly inhibit the tumorigenic activity of hESCs, but do not affect their versatility.

Accordingly, in one aspect, the invention provides a method of removing undifferentiated pluripotent stem cells from a sample containing such cells, the method comprising exposing the sample to an effective amount of a cardiac glycoside.

In some embodiments, the cardiac glycoside used herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein L is a lactone group;

R₁、R₄and R₅Independently is H or OH;

R₂is CH₃Or CH₂OH；

R₃H or OH in the absence of the dotted line, or R₃Is absent when the dotted line forms a double bond;

R₄is H or OH;

R₅is H or OH;

R₆is CH₃(ii) a And

x is-OH or a glycoside of 1 to 6 sugar residues, each unsubstituted or substituted.

In some embodiments, the lactone group is an unsaturated, five or six membered lactone ring.

In some embodiments, the sugar residue is selected from the group consisting of rhamnose, glucose, digitoxin, digitose, glucoronise, digitose (digginose), rhabdomyosyl (sarmentose), valarose (vallarose), and fructose.

In some embodiments, the sugar residue is unsubstituted or substituted with an acyl group, an amine group, a halogen group, and/or an amide group.

In certain embodiments, the compound of formula (I) is selected from the group consisting of:

and

in some embodiments, the undifferentiated pluripotent stem cells are selected from the group consisting of Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs).

In some embodiments, the undifferentiated pluripotent stem cells exhibit a cellular marker selected from the group consisting of Na⁺/K⁺-ATPase, Nanog, Oct4, Sox2, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and combinations thereof.

In some embodiments, the sample further comprises differentiated cells.

In some embodiments, the method further comprises culturing the differentiated cells to provide a population of the differentiated cells.

In some embodiments, the differentiated cells are Mesenchymal Stem Cells (MSCs). In particular, MSCs displayed a cellular marker selected from the group consisting of CD44⁺、CD73⁺、CD90⁺、CD105⁺And combinations thereof, and typically CD45^-、CD34^-、CD11b^-、CD19^-And/or HLA-DR^-。

In some embodiments, the differentiated cells are selected from the group consisting of osteoblasts, adipocytes, chondrocytes, endothelial cells, neural cells, and hepatocytes.

In another aspect, the present invention provides a method for preparing a differentiated cell, comprising:

(i) culturing undifferentiated pluripotent stem cells under conditions suitable for differentiation to produce a cell population comprising differentiated cells and undifferentiated pluripotent stem cells;

(ii) exposing the cell population to an effective amount of a cardiac glycoside to remove undifferentiated pluripotent stem cells; and

(iii) the remaining differentiated cells may be cultured as necessary.

In some embodiments, the cardiac glycoside used herein is a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof.

In yet another aspect, the invention provides a method for treating a subject in need of cell therapy, comprising administering to the subject a population of cells comprising differentiated cells, wherein the population of cells is exposed to a cardiac glycoside prior to administration to the subject, thereby removing, if any, undifferentiated cells present in the population of cells. In some embodiments, the cardiac glycoside used herein is a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof.

Also provided herein is the use of a cardiac glycoside for the manufacture of a composition for inducing cell death of and/or removing undifferentiated pluripotent stem cells from differentiated cells. Further provided are compositions for cell therapy comprising a cardiac glycoside and a population of cells comprising differentiated cells. In some embodiments, the cardiac glycoside used herein is a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof.

Further provided are methods for treating a teratoma in a subject in need thereof comprising administering to the subject an effective amount of a cardiac glycoside.

The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will become apparent from the following detailed description of several specific embodiments, and from the claims.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

In the following figures:

fig. 1A-1E include graphs showing that cardiac glycosides induce cytotoxic effects in hESCs but not in hMSCs. FIG. 1A shows Na in hESCs and hMSCs detected by Western blotting⁺/K⁺hESCs and hMSCs were treated with DMSO solvent control, 2.5 μ M digitonin, or 2.5 μ M digitonin C (12 hours and 24 hours). Scale: 500 μ M. FIGS. 1C and 1E show LDH release measured in hESCs and hMSCs to study cytotoxic effects in 96-well dishes, hESCs or hMSCs were treated with DMSO, 2.5 μ M digitonin, or 2.5 μ M digitonin C for 24 hours, and supernatants were collected for testing<0.001；**P<0.01; n.s.: has no significance. Data are shown as mean ± SD.

Fig. 2A-2D include graphs showing that cell death markers are upregulated in cardiac glycoside-treated hESCs but not in hBMMSCs. Cell death was analyzed as hESCs or MSCs. FIG. 2A shows hESCs treated with DMSO, 1.25 μ M digitonin, or 2.5 μ M ouabain C for 24 hours. The bar graph represents statistics of flow cytometry data. P<0.05; data are shown as mean ± SD. FIG. 2B shows hMSCs treated with DMSO, 2.5 μ M digitonin, or 2.5 μ M ouabain C for 24 hours. Cells were stained with PI and annexin V and analyzed by flow cytometry. The living cells are PI^-Annexin V^-Represents; the dead cells are PI^-Annexin V⁺、PI⁺Annexin^-And PI⁺Annexin⁺And (4) showing. The bar graph represents statistics of the FASC data. n.s.:has no significance. Data are shown as mean ± SD. FIGS. 2C and 2D show the Western blot method for detecting the amount of disrupted proteins in the apoptotic markers and the universal stem cell markers. hESCs and hMSCs were treated with DMSO, 2.5. mu.M digitonin, or 2.5. mu.M ouabain C for 12 hours. Cells were harvested and tested for cleaved and uncleaved PARP, apoptotic protease 7, and apoptotic protease 3. To detect the marker of the universal stem cells, the cells were harvested and Nanog and Oct4 were detected.

Fig. 3A-3C include graphs showing that treatment of hBMMSCs with cardiac glycosides did not affect their differentiation ability. hBMMSCs were treated with DMSO, 2.5 μ M digitonin, or 2.5 μ M ouabain C for 24 hours and the drug was removed for further differentiation. Fig. 3A shows osteogenic differentiation. Mineralization was stained with alizarin Red s (alizarin Red s) and quantified at o.d 570 nm. Figure 3B shows adipogenic differentiation. The lipid droplets were stained with oil red and quantified at o.d 510nm. Figure 3C shows chondrogenic differentiation. Glycosaminoglycans were stained with alician blue (Alcian blue) and quantified at o.d 650 nm. Scale bar: 500 μm. n.s.: has no significance. Data are shown as mean ± SD.

Fig. 4A-4E include graphs showing that cardiac glycosides do not affect the viability of H9 hESC-derived MSCs cells. FIG. 4A shows the cell morphology of H9hESC-MSCs in brightfield. hESC-MSCs were treated with DMSO solvent controls, 2.5. mu.M digitonin, or 2.5. mu.M ouabain C for 12 hours and 24 hours, respectively. Scale bar: 500 μm. Figure 4B shows the measured release of LDH in hESC-MSCs to study cytotoxic effects in 96-well culture discs. Cells were treated with DMSO or drug for 24 hours, after which the supernatant was harvested for LDH detection. Samples were normalized to DMSO-treated hESCs. Figure 4C shows flow cytometry for cell death analysis. hESC-MSCs were treated with DMSO, 2.5. mu.M digitonin, or 2.5. mu.M ouabain C for 24 hours. Cells were stained with PI and annexin V and analyzed using flow cytometry. The bar graph represents statistics of flow cytometry data. n.s.: has no significance. Data are shown as mean ± SD. FIG. 4D shows hESCs and hESC-MSCs treated with DMSO, 2.5 μ M digitonin, or 2.5 μ M ouabain C for 12 hours. Cells were harvested and cleaved and uncleaved PARP, apoptotic protease 3, and apoptotic protease 7 were detected using western blot.

FIG. 4E shows Western blot detection of Na in hESCs and hESC-MSCs⁺/K⁺-protein expression of ATPase α 1 and α 2 subunits.

Fig. 5A-5C include graphs showing that treatment with cardiac glycosides can prevent teratoma formation in NSG mouse hESCs two months (groups n-8) scale bar: 10mm, fig. 5A shows this graph, fig. 5B shows quantification of teratoma weight, point: DMSO, square: digitoxin, triangle: hairy glycosides C, P < 0.0001. data are shown as mean ± SD, fig. 5C shows images of their DMSO treated hESCs derived teratomas paraffin sections stained with H & E stain (top) versus IHC stain (bottom) lineage AFP (α fetoprotein) marker SMA (smooth muscle actin): mesomarker.tuj 1(β -III protein): microtubule 50 μm scale bar.

Fig. 6A-6B include graphs showing that cardiac glycosides cause HUES6 hESCs cytotoxicity. Fig. 6A shows cell colonies and morphology of HUES6 in bright field. S6 hESCs were treated with DMSO, 2.5. mu.M digitonin, or 2.5. mu.M ouabain C for 12 hours and 24 hours. The cells become round and die. FIG. 6B shows S6 hESCs treated with DMSO, 2.5. mu.M digitonin, or 2.5. mu.M ouabain C for 24 hours. Culture supernatants were harvested for LDH release detection. P < 0.001. Data are shown as mean ± SD.

Figure 7 shows that the bufadienolide subpopulations of cardiac glycosides, bufalin and prodelphinogen a, induced cytotoxic effects in hESCs without affecting hBMMSCs. hESCs (top panel) and hBMMSCs (bottom panel) were treated with different cardiac glycosides, respectively, at a final concentration of 2.5 μ M for 24 hours. The cytotoxic effect was determined using the LDH release assay and all data were compared to the DMSO solvent control group. Cardiac glycosides: digitonin and ouabain C; toad cardiac glycosides: bufalin and prodelphinidin A. P < 0.0001; n.s.: has no significance. Data are shown as mean ± SD.

Fig. 8A to 8D include graphs showing the results of cytotoxicity assays. FIG. 8A shows a schematic of the differentiation process, and MSCs were derived from H9 hESCs. FIG. 8B shows the 1 st generation hESC-MSCs phenotype analyzed and sorted with CD73 and CD 105. Fig. 8C shows the general MSC positive and MSC negative markers analyzed in hESC-MSCs. Positive marker: CD73, CD90, CD44, and CD 105; negative marker: CD45, CD34, CD11b, CD19, and HLA-DR. FIG. 8D shows hESC-MSCs treated with different cardiac glycosides at 2.5 μ M final concentrations for 24 hours, respectively, before measurement of LDH release in cytotoxicity assays. n.s.: has no significance. Data are shown as mean ± SD.

FIGS. 9A-9G include graphs showing that cardiac glycosides do not or slightly affect hPSC-derived cell survival, FIG. 9A shows FASC analysis of endothelial cell markers CD34 and CD144, which shows that there is more than 90% of the CD34+/CD144+ population in the hipSC endothelial cells, FIGS. 9B and 9C show the results of cytotoxicity assays in which cells are treated with 2.5 μ M epirubicin and 2.5 μ M epirubicin C for 24 hours, and the amount of LDH release determined in the cytotoxicity assays, cardiac glycosides induce cytotoxicity in undifferentiated hipSCs, but do not affect the viability of hipSC-endothelial cells, FIG. 9D shows immunofluorescence staining of the neuron marker TUJ1 in the heSC-neurons, Green: TUJ1, DAPI (blue colored). FIG. 9E shows that heSC-neurons are treated with 2.5 μ M epirubicin and 2.5 μ M epirubicin C for 24 hours, and the amount of LDH-endoderm labeled with 2.5 μ M epirubicin C determined in terms of blue nuclear fluorescence, FIG. 9E shows DAPI (blue nuclear fluorescence staining of the cells) and AFP-cells in the cell nuclear protein.

FIG. 9G shows that hESC-hepatocyte endoderm cells were treated with 2.5 μ M digitonin and 2.5 μ M digitonin C for 24 hours and slightly induced cytotoxicity in terms of measured LDH release. Scale bar: 200 μm. P < 0.01; p < 0.05; n.s.: has no significance. Data are shown as mean ± SD.

Fig. 10 shows immunohistochemical images of cardiac glycoside treated teratomas, these images representing paraffin sections showing three germ layer lineages of cardiac glycoside treated teratomas sections of digoxigenin treated teratomas were stained with H & E (top panel) and IHC staining was performed for three lineage markers (bottom panel). sections of trichoroside C treated teratomas were stained with H & E (top panel) and IHC staining was performed for three lineage markers (bottom panel). AFP (α -fetoprotein): endoderm marker.sma (smooth muscle actin): mesodermal marker.tuj 1(β -III tubulin): ectodermal marker.scale: 50 μm.

Fig. 11A-11C include graphs showing that drug-treated hBMMSCs remain in NSG mice after cell transplantation. FIG. 11A shows that hBMMSCs overexpressing GFP were injected subcapsularly and stained with GFP antibody to demonstrate that the cells remained in NSG mice. Fig. 11B shows the left image: NSG mice kidney subcapsular teratoma sections from a mixture of undifferentiated hESCs and hBMMSCs overexpressing GFP, both treated with DMSO, digitonin, or piloside C; and right drawing: the tumor area of the kidney tissue was quantified using image display software (n-3). Scale bar: 2 mm. Fig. 11C shows teratoma section staining with GFP antibody (top panel) and H & E (bottom panel), showing that drug-treated hBMMSCs remain at the graft site. Scale bar: 50 μm.

Figure 12 shows that cardiac glycosides (digitonin, ouabain C, prosaposin a, digitoxin, ouabain) induce cytotoxic effects in hESCs in a dose-dependent manner.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. ""

1. Definition of

As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a component" includes a plurality of such components and equivalents thereof known to those skilled in the art.

The terms "comprises," "comprising," or the like, are generally used in an inclusive/sense, meaning that one or more features, ingredients, or components are allowed to be present. The words "comprising" or "containing" and the like encompass the words "consisting of" or "consisting of … and the like.

The term "undifferentiated pluripotent stem cells" as used herein means cells that are capable of self-renewal and pluripotent. The term "universal" means the ability of a cell to differentiate into all cell lines except the placenta. In particular, universal cells include those that can differentiate into the three major germ layers (germ layers), i.e., endoderm, ectoderm, and mesoderm. Generally, undifferentiated pluripotent stem cells are Embryonic Stem Cells (ESCs), which may be derived from embryonic sources, such as pre-embryonic, or fetal tissues at any time after fertilization. Undifferentiated pluripotent stem cells may also include Induced Pluripotent Stem Cells (IPSCs) artificially derived from non-pluripotent cells (e.g., somatic cells) by insertion of one or more specific genes or stimulation with chemicals. Induced pluripotent stem cells are considered to be the same as pluripotent stem cells (e.g., embryonic stem cells) because they have two unique features, namely self-renewal capacity and versatility. ESCs or IPSCs can form teratomas. ESCs or IPSCs are more known to express specific cellular markers, such as Na⁺/K⁺-ATPase, Nanog, Oct4, Sox2, SSEA3, SSEA4, TRA-1-60, TRA-1-81, alkaline phosphatase.

As used herein, "mesenchymal stromal/stem cells (MSCs)" are self-renewing and pluripotent (multipotent) the term "pluripotent" as used herein means that stem cells have the ability to differentiate into multiple cell types, pluripotent stem cells are not capable of producing any type of mature cells in vivo, are limited to a limited range of cell types⁺、CD73⁺、CD90⁺、CD105⁺And is CD45^-、CD34^-、CD11b^-、CD19^-And/or HLA-DR^-。

The term "differentiation" as used herein refers to the process of differentiation of a universal stem cell into progeny, wherein the cell is enriched for a particular form or function. Differentiation is a relative process. For example, MSCs are relatively differentiated compared to ESCs, but still have the ability to differentiate into multiple cell types. Mature somatic cells, such as osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat), hepatocytes (liver) may be in a final differentiated form, which loses the ability to differentiate into different cell types. Thus, the term "differentiated cell" may refer to a relatively differentiated cell, such as MSCs, or a terminally differentiated cell, such as a mature somatic cell.

As used herein, the terms "remove" or "eliminate" when used in relation to undifferentiated pluripotent stem cells mean to separate or isolate such cells from other components in the original sample or components left in the sample after one or more processing steps. Other components may include, for example, other cells, particularly differentiated cells. Removing or eliminating the target cells can include killing, inhibiting, or depleting the target cells in the sample by applying the compounds used herein, for example, such that the sample will be enriched for other components, such as differentiated cells. Killing the target cell may include inducing apoptosis or cytotoxicity. Inhibiting or depleting a target cell can include reducing the number, ratio, proliferation, or activity (universal capability or tumorigenic activity) using a measurable amount. The removal may be partial or complete. As used herein, a sample or culture is substantially free of undifferentiated pluripotent stem cells, for example, may contain less than about 10%, about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or unmeasured undifferentiated pluripotent stem cells.

The term "culture" as used herein refers to a population of cells cultured in a medium. The cells can be subcultured (passage). The cell culture may be a primary culture, which is not subcultured after isolation from animal tissue, or may be subcultured multiple times (subculture one or more times).

The term "about" as used herein means plus or minus 5% of the numerical value of the number used.

The term "subject" as used herein includes humans and non-human animals, such as companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like), or laboratory animals (e.g., rats, mice, guinea pigs, and the like).

The term "treatment" as used herein refers to the application or administration of a composition comprising one or more active agents to a subject having a disease, symptom or condition of a disease, or disease progression with the purpose of treating, curing, alleviating, altering, remediating, ameliorating, improving, or affecting the disease, symptom or condition of a disease, disability caused by a disease, or disease progression.

The term "effective amount" as used herein means the amount of active ingredient that confers a biological effect on the treated cells or individuals. The effective amount may vary depending on various reasons, such as the route and frequency of treatment, body weight, and cell type or individual receiving the active ingredient.

As used herein, "lactone" groups are cyclic esters. In particular, the lactone may be represented by the formula:

wherein R is¹And R²Together with the carbon and oxygen atoms to which they are attached form an optionally substituted cyclic group, which may be saturated, unsaturated, or aromatic. R¹And R²Independently hydrogen or an optionally substituted hydrocarbon moiety. The most stable structures of lactones are 5-membered gamma-lactones and 6-membered delta-lactones.

"hydrocarbon" refers to an organic compound consisting of hydrogen and carbon, which may be linear, branched, or cyclic, and may include alkanes, alkenes, alkynes, aromatics, or combinations thereof.

The term "glycoside" refers to a compound in which a sugar group or sugar residue is bound to a non-carbohydrate moiety. Typically, a sugar group (glycone) is bonded through its first isomeric carbon (anomeric carbon) to another group (aglycone) via a glycosidic bond and having an oxygen, nitrogen, or sulfur atom as a linker.

The sugar group or sugar residue used herein may be a simple sugar, an amino sugar, a deoxy sugar, a sugar acid, or a sugar alcohol.

The term "optionally substituted" as used herein means that the group to which the term refers may be unsubstituted or may be substituted with one or more substituents. Examples of the substituent may include an alkyl group, an alkoxy group, a hydroxyl group, an acyl group, a halogen group, and/or an amide group. Examples of the alkyl group include C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl. Examples of the alkoxy group include C1-6 alkoxy groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, and tert-butoxy. Examples of acyl groups include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, hexanoyl, octanoyl, decanoyl, lauroyl, and benzoyl. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

The term "pharmaceutically acceptable salts" as used herein includes acid addition salts. By "pharmaceutically acceptable acid addition salts" is meant those salts which retain the biological effectiveness and properties of the free base and are formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, pyruvic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, trifluoroacetic acid, and the like.

2. Compound (I)

According to the invention, the active compound used herein is a cardiac glycoside.

Cardiac glycosides, as used herein, are inhibitors of the cell membrane Na +/K + ATPase (ATP phosphohydrolase) —. in particular, cardiac glycosides comprise a steroid core with a pyrone (pyrone) or butyrolactone substituent at the C17 position ("pyrone form" versus "butyrolactone form"), and typically the C3 position is glycosidated (glycosylated) — most cardiac glycosides comprise one to four sugars attached to the 3 β -OH group.

In some embodiments, the cardiac glycoside is a compound represented by formula (I):

and pharmaceutically acceptable salts thereof,

wherein L is a lactone group;

R₁、R₄and R₅Independently is H or OH;

R₂is CH₃Or CH₂OH；

R₄is H or OH;

R₅is H or OH;

R₆is CH₃(ii) a And

In some embodiments, the sugar residue is selected from the group consisting of rhamnose, glucose, digitoxin, digitose, lowgo, cladosporium vinium, malasse, and fructose.

In some embodiments, X is a glycoside of 1, 2, 3, 4,5, or 6 sugar residues. In some embodiments, X is a glycoside of two parts digitoxin, one part acetyl digitoxin, and one part glucose; or X is the glycoside of the digitoxin; or X is a portion of rhamnose glycoside.

In some embodiments, the sugar residue is unsubstituted' or substituted with an acyl group, an amine group, an acetyl group, a halogen group, and/or an amide group.

Examples of cardiac glycosides include, but are not limited to, digitoxin (digoxin), verbascoside C (lantoside C), barbascoside (ouxin), sanocidin A (angiotoxin), echinocandin (saponin A), sanocidin (saponin C), carissoside (saponin A), carissoside (saponin C), carissoside (saponin A), carissoside (saponin C-saponin C), carissoside (saponin E, saponin E-saponin A), carissoside (saponin C-saponin E, saponin E (saponin E-D), carissoside (saponin A), carissoside (saponin E-D), carissoside (saponin A), echinocandin-saponin A), echinocandin (saponin A), echinocandin-D (saponin E) (saponin C), echinocandin-D), echinocandin (saponin E (saponin E) (saponin C), echinocandin-D), echinocandin-saponin E (saponin E) (saponin C), echinocandin-D), echinocandin (saponin E) (saponin E-D), echinocandin (saponin (D), echinocandin (saponin E) (saponin), echinocandin (saponin E) (saponin E-E) (saponin C), echinocandin (E) (saponin C), echinocandin (I) (saponin (E) (saponin C), echinocandin (I) (saponin (I), echinocandin (I) (saponin C), E) (saponin (I), echinocandin (E) (saponin (I) (saponin C), echinocandin (I) (saponin (E) (saponin C), E) (saponin (E) (saponin C) (saponin (E) (saponin C), echinocandin (E) (saponin C) (saponin (E) (saponin C), echinocandin (E) (saponin C), orisin), E) (saponin C) (saponin (E) (saponin C), E) (saponin (I) (saponin C) (saponin (E) (saponin C), E) (saponin C) (saponin (I), E) (saponin C) (saponin (E) (saponin C) (saponin (E) (saponin C) (saponin (E) (saponin C), E) (saponin (I), saponin C) (saponin (E) (saponin (I), saponin C), E) (saponin C) (saponin (I), E) (saponin C), saponin C) (saponin C), E) (saponin C) (saponin (E) (saponin C) (saponin (E) (saponin C) (saponin (E) (saponin C), E) (saponin C), saponin C) (saponin C) (saponin (I), saponin C) (saponin (E) (saponin C) (saponin (I) (saponin C) (saponin (E) (saponin C) (saponin (E) (saponin C) (saponin (E) (saponin C) (saponin), saponin (E) (saponin C) (saponin), saponin C) (saponin C) (saponin (E) (saponin), saponin (E) (saponin), saponin (E) (saponin), saponin (saponin), saponin (E) (saponin (E), saponin C) (saponin (E) (saponin C) (saponin), saponin (E) (saponin C) (saponin E) (saponin (E) (saponin (.

In some examples, the compound is selected from the group consisting of

And

the compounds of the present invention may be prepared using conventional methods or may be purchased commercially.

3. Use of active compounds

According to the present invention, cardiac glycoside can be used to induce cell death of undifferentiated pluripotent stem cells, and thus undifferentiated pluripotent stem cells can be selectively removed from differentiated cells.

In particular, the invention provides a method of removing undifferentiated pluripotent stem cells from a sample containing such cells, comprising exposing the sample to an effective amount of a cardiac glycoside.

In some embodiments, the sample is a cell sample comprising cultured cells (in vitro) to be transferred to a patient in need of cell therapy.

Specifically, the present invention also provides a method of preparing a differentiated cell, comprising:

(ii) exposing the population of cells to an effective amount of a cardiac glycoside to remove the undifferentiated pluripotent stem cells; and

(iii) the remaining differentiated cells may be cultured as necessary.

In some embodiments, the undifferentiated pluripotent stem cells are selected from the group consisting of Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs). Preferably, the pluripotent stem cells are derived from a human. Human ESCs can be obtained from human blastocyst cells using techniques well known in the art. Human IPSCs can be prepared by isolation and culture of suitable somatic donor cells (e.g., human fibroblasts) and genetically engineered using techniques well known in the art.

In some embodiments, the undifferentiated pluripotent stem cells are cultured in a medium under conditions that allow a portion of the undifferentiated pluripotent stem cells to differentiate into differentiated cells of interest, followed by the addition of an effective amount of a cardiac glycoside to the medium such that the remaining undifferentiated pluripotent stem cells die, and optionally the remaining differentiated cells are further culture expanded and then administered to an individual in need of cell therapy.

In some embodiments, suitable media for culturing the undifferentiated pluripotent stem cells and/or differentiated cells of the invention are available in the art, such as DMEM, MEM, or IMEM media, with 10% fetal bovine serum. The cell culture can be performed under normal conditions, e.g., 1-5% CO at 37 ℃₂Under the conditions. Typically, an appropriate amount of cardiac glycoside is added to the medium to a concentration of 0.1 μ M to 100 μ M, after which the cells are cultured in the presence of the cardiac glycoside for at least 3 hours, at least 6 hours, preferably 12 hours, more preferably 24 hours, to allow the remaining undifferentiated pluripotent stem cells to die. Media components may be added to promote differentiation, which promotes differentiation into the desired cell line (cell line).

By treatment with a cardiac glycoside, the remaining undifferentiated pluripotent stem cells can be selectively killed and removed from their differentiated progeny, so that the sample containing the differentiated progeny and from which the undifferentiated pluripotent stem cells have been removed can be used for cell therapy with reduced risk of developing tumors (tomogenic risk). In particular, the amount of viable undifferentiated pluripotent stem cells after treatment with cardiac glycoside is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than a control (e.g., the same cells not so treated). More specifically, the removal action is complete; that is, undifferentiated pluripotent stem cells were completely dead after this treatment, and the remaining undifferentiated pluripotent stem cells were not detected.

Accordingly, the present invention further provides a method for treating a subject in need of cell therapy comprising administering to the subject an effective amount of a cell population comprising differentiated cells, wherein the cell population is exposed to a cardiac glycoside prior to administration to the subject, thereby substantially removing, if any, undifferentiated pluripotent cells present in the cell population. Typically, the cell population is further replaced with fresh medium that is free of cardiac glycoside such that the cell population is free of cardiac glycoside prior to administration to the subject.

Also provided is a composition comprising a cardiac glycoside and a cell population comprising differentiated cells. In particular, the population of cells has been exposed to a cardiac glycoside prior to administration to the subject, thereby substantially removing, if any, undifferentiated cells present in the population of cells.

Typically, for purposes of delivery and efficacy, the compositions are formulated in a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition and preferably stabilizes the active ingredient, as well as being safe for the person receiving the treatment. The carrier may be a diluent, carrier, excipient, or matrix for the active ingredient. The compositions of the present invention may be delivered via any physiologically acceptable route, such as oral, parenteral (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal routes. For parenteral administration, a preferred form of use is a sterile aqueous solution which may contain other substances sufficient to make the solution isotonic with blood, such as salts or glucose. The aqueous solution may be suitably buffered as required.

In some embodiments, the subject in need of cell therapy is suffering from a disease or condition, including, but not limited to, macular degeneration (macular degeneration), graft-versus-host disease, cardiac disease (e.g., peripheral arterial disease, ischemia, stroke, myocardial infarction), Acute Lung Injury (ALI), Crohn's disease, type i diabetes, multiple sclerosis, neurological disease, osteogenesis imperfecta (osteoprogenitor), ischemia, fibrosis, and genetic disease, such as Hurler's syndrome, anti-inflammatory response (immunoregulatory), cardiovascular disease, neurodegenerative disease, tissue engineering, and the like. The treatment may use the cells to construct new tissue (with or without biological material) according to any method well known in the art. The cells may be injected or transplanted to the site of tissue damage so that they create new tissue in vivo. For example, MSCs may be used in cartilage and bone regeneration for the treatment of arthritis, Lower Back Pain (LBP), cartilage degradation, bone fracture, or osteoporosis. In addition, since MSCs are classified into fat and cartilage, MSCs can also be applied to plastic surgery, such as cartilage transplantation in autologous fat transplantation surgery and nasal augmentation surgery.

The invention will be further illustrated by the following examples, which are provided for purposes of illustration and not limitation. It will be appreciated by those of ordinary skill in the art that, in light of the present disclosure, many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

An important safety concern with the use of human pluripotent stem cells (hPSCs) is the risk of tumorigenesis, since these cells can form teratomas at ectopic sites after in vivo injection. Thousands of undifferentiated hPSCs were sufficient to induce teratomas in a mouse model. Therefore, it is crucial to remove all residual undifferentiated hPSCs with teratoma potential before clinical application of hPSC-derived cells. In this study, the inventors' data demonstrated the cytotoxic effects of cardiac glycosides, such as digitonin, floroside C, bufalin, prodelphinogen A, digitoxin, and ouabain, in undifferentiated human embryonic stem cells (hESCs). This was not observed in human mesenchymal stem cells (hBMMSCs). Most importantly, digitonin and ouabain C do not affect the differentiation ability of stem cells. Consistently, the viability of hESC-derived MSCs, neurons, and endothelial cells, hepatocyte endoderm were not or slightly affected by treatment with digitonin and ouabain C. In addition, in vivo experiments prove that the digitonin and the hairy flower glycoside C can prevent the formation of teratoma. This is the first description of the cytotoxic and tumor preventative effects of cardiac glycosides in hESCs according to the present invention. Digitonin and ouabain C are also the first FDA-approved drugs with cytotoxicity in undifferentiated hESCs.

1. Materials and methods

All methods are performed in accordance with relevant guidelines and regulations. All experiments were approved by the Central institute of technology (Taipei, Taiwan) human Individual research ethics Committee (AS-IRB02-106069) and the institutional animal Care and use Committee (IACUC, 14-03-684). Unless otherwise indicated, all media and supplements were purchased from ThermoFisher scientific (Wilmington, DE, USA). All chemicals were purchased from Sigma (st. louis, MO, USA) unless otherwise noted.

1.1 cell lines and culture conditions

hESC cell line H9 was purchased from WiCells (Madison, Wis., USA)²⁷. Another hESC cell line, HUES6, was donated from Dr. Douglas A. Melton (Harvard University, Boston, MA, USA)²⁸. In the feeder (feeder) cell culture, Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 20% knockout serum replacement, 2 mML-glutaminic acid, 1% non-essential amino acids, 4ng/mL human bFGF, and 0.1mM 2-mercaptoethanol was used for culture. In terms of feeder cell culture, C57BL/6 Mouse Embryonic Fibroblasts (MEFs) were cultured in DMEM containing 10% FBS and treated with 0.01mg/ml mitomycin C for 2-6 hours to inactivate the cells. For feeder cells-free culture, hESCs were seeded on culture plates coated with matrigel matrix (BD Biosciences, San Jose, CA, USA) and cultured with MEF (C57BL/6) conditioned medium. Culturing hBMMSCs in mesenPRO RS^TMIn a kit (Life Technologies, Camarillo, Calif., USA). All cells were cultured in the presence of 5% CO₂At 37 ℃ in a humid environment.

1.2 Lactate Dehydrogenase (LDH) cytotoxicity assay

Cell supernatants were harvested that were treated with digitonin 2.5 μ M, ouabain C2.5 μ M, or DMSO solvent controls for 24 hours. The released LDH was determined by CytoTox96 non-cytotoxic assay according to the manufacturer's instructions (Promega, Southampton, UK). The supernatant was incubated with the reagents at room temperature for 20 minutes, after which the reaction was stopped with a stop solution. The absorbance at 490nm was measured using a read-disc Spectrophotometer (Benchmark Plus Microplate Spectrophotometer System, BIO-RAD, CA, USA).

1.3 Western blot analysis

Cell lysates were harvested using RIPA buffer (RIPA buffer: NaCl 150mM, Tris pH 8.050 mM, EDTA pH 8.05 mM, NP-401.0%, SDS 0.5%, sodium deoxycholate 0.1%). As previously described, in a different wayWestern blot analysis of primary antibodies of type²⁹Primary antibodies used included anti- β actin (A5441; Sigma), anti-Oct 4 (sc-9081; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Nanog (3580; Cell Signaling Technology, Danvers, MA, USA), apoptosis antibody kit (9915; Cell Signaling Technology), ATP1A1 (3010; Cell Signaling Technology), and ATP1A2 (16836-1-AP; proteintech Technology)^TM). After reaction at4 ℃ overnight, the blots were cultured in goat anti-mouse or goat anti-rabbit antibodies conjugated with horseradish peroxidase (jackson immunoresearch Inc). The blots were detected using a chemiluminescent substrate (WBKLS 0500; Millipore, Darmstadt, Germany). Images were taken using Fujifilm LAS-4000(FUJIFILM, Tokyo, Japan).

1.4 flow cytometry

In the cell death assay, the manufacturer's instructions (Alexa)

488 annexin V/dead cell apoptosis kit; life Technologies) were performed on the propidium iodide/annexin V assay. Briefly, live cells were detached with trypsin and incubated with annexin V antibody and PI working solution for 15 minutes. The inventors added 400. mu.l of 1X annexin binding buffer and used FACSCAnto^TM(Becton Dickinson, Franklin Lakes, NJ, USA) stained cells were analyzed. In analyzing the phenotypic characteristics of MSCs, the inventors used Stemflow^TMhMSC assay kit (BD Biosciences) and FACSCAnto^TM. All flow data were generated using FACSDiva^TMSoftware (BD Biosciences) and FlowJo^TM(FlowJo, LLC, Ashland, USA).

1.5 hESC-derived MSCs

Xu and co-workers' reports under dr³⁰hESCs were cultured with 10ng/ml BMP4 and 1. mu.M A8301 for 5 days, then the cells were passaged on gelatin-coated plates and the medium was switched to MSC medium [ minimal essential medium Eagle α modified (α MEM) medium supplemented with 20% Fetal Bovine Serum (FBS), L-glutamic acid (Gluta-MAX), 1X non-essential amino acids]. The cells are inAmplification within passage 5 and sorting out CD73⁺(11-0793, ThermoFisher scientific) and CD105⁺(12-1057, ThermoFisher Scientific) double positive cells.

1.6 cell sorting

hESC-derived MSCs were detached with trypsin and washed with PBS. Then, the cells were incubated with anti-human CD73FITC (ThermoFisher Scientific) and anti-human CD105 PE (ThermoFisher Scientific) at4 ℃ for 15 minutes. Cells were washed three times with PBS. Sorting CD73 with cell sorter (BD FACSAria II, BD Biosciences)⁺/CD105⁺A cell. The sorted cells were cultured in MSC medium.

1.7 osteogenic differentiation and alizarin Red S (Alizarin Red S) staining

BMMSCs were treated with digitonin (2.5. mu.M), ouabain C (2.5. mu.M), or DMSO solvent controls for 24 hours. The drug was removed and the cells were washed with PBS and cultured in MSCgo^TMOsteogenic differentiation medium (biologicalcalel industries, Kibbutz Beit-Haemek, Israel)³¹. The medium was changed twice a week for 14-21 days. Subsequently, the cells were fixed with ice-cold 70% ethanol at-20 ℃ for 1 hour. After washing three times with water, the cells were subsequently stained with 40mM Alizarin Red S (ARS) (pH 4.2) for 10 minutes at room temperature. Next, the cells were washed five times with PBS. Images were taken using an Olympus CK-40 microscope. In terms of quantification, the stain was extracted with sodium phosphate buffer (pH 7.0) containing 10% (w/v) cetylpyridinium chloride (Sigma 0732) for 15 minutes and the o.d. value at 570nm was determined.

1.8 lipogenesis and oil Red O test

hBMMSCs were treated with digitonin (2.5 μ M), ouabain C (2.5 μ M), or DMSO solvent controls for 24 hours. Subsequently, the drug was removed and the cells were cultured in MSCgo^TMAdipogenic differentiation medium (biologicalcalel industries). The medium was changed every 3-4 days for 8-12 days. After induction of adipogenesis, the inventors replaced MSCgo with MSC maintenance medium^TMComplete medium for adipogenesis for 6-9 days. Cells were carefully fixed with 4% formaldehyde for 1 hour, washed with 60% isopropanol, and air dried at room temperature. Fat droplets (lipid drops) in oil redO operating solution (30ml of 0.35% oil red solution in isopropanol, diluted with 20ml of distilled water) was stained for 10 minutes. Subsequently, the cells were washed 4 times with water. In terms of quantification, the oil red O stain was extracted with 100% isopropanol and the absorbance at 510nm was measured.

1.9 cartilage induction (chondrogenic induction) and Elson Blue (Alcian Blue) assays

BMMSCs were treated with digitonin (2.5. mu.M), ouabain C (2.5. mu.M), or DMSO solvent controls for 24 hours. Next, the drug was removed and 1x 10 was added⁵The individual cells were seeded in 96-well U-bottom culture plates. After 24 hours, the inventors changed to complete MSCgo^TMCartilage induction medium (Biological Industries) for 21 days. The medium was changed every 3-4 days. Next, the pelleted cells were fixed with 4% formaldehyde for 1 hour, and ddH was added₂O washes twice and stains with 0.1N HCl in 1% aixon blue for 30 minutes. For the elution of albson blue (elution), the inventors added 8M guanidine hydrochloric acid solution and incubated at room temperature overnight. The absorbance at 650nm was measured.

1.10 in vivo tumorigenic assays and immunohistochemistry

hESCs were treated with digitonin (2.5 μ M), floridin C (2.5 μ M), or DMSO controls for 24 hours. About 10 in 50. mu.l PBS⁶Each treated cell was combined with 10⁵The MEFs are mixed to promote teratoma formation³². The cell mixture was mixed well with 1x Matrigel Matrix and cells were injected subcutaneously into NOD scid γ mice (NSG mice) for 8 weeks. After 8 weeks, animals were sacrificed and teratomas were removed, fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. H&The E staining method was modified based on previous studies³³In immunohistochemistry, teratoma sections were blocked with 5% milk for 1 hour and stained with primary antibody overnight at4 ℃, followed by treatment with secondary antibody (Dako, Santa Clara, CA, USA) for 1 hour at room temperature, and DAB enhancer (Dako). primary antibody anti-human α -1-fetoprotein for endoderm lineage (a0008, Dako), anti-human smooth muscle actin for mesoderm lineage, germline strain 1a4(M0851, Dako); with secondary antibodyanti-Tuj 1 in the ectoderm (MAB1637, EMD Millipore, Darmstadt, Germany).

1.11 differentiation of hiPSC cells into endothelial cells

hiPSC-derived endothelial cells were prepared according to previous reports³⁴. Hipscs were maintained in mTeSR1 medium (stem cell Technologies, Vancouver, Canada) on VTN-N coated culture plates (Thermo, Wilmington, DE, USA). First, to induce differentiation stepwise, the inventors induced mesoderm of hiPSCs grown in mesoderm-inducing medium for 48 hours (basal medium containing 10mM Y-27632, 3mM CHIR99021, and 2ng/ml activin A; basal medium consisting of 12g/L DMEM/F-12, 3.56g/L HEPES, 1.742g/L sodium bicarbonate, 14. mu.g/L sodium selenite, 10.7mg/L recombinant transferrin, 19.4mg/L recombinant insulin, and 64 mg/L-ascorbic acid 2-sesquihydrate of magnesium sesquiphosphate). Again, for mesodermal to endothelial cell conversion, the mesodermal cells were replanted in angiogenesis medium (basal medium supplemented with 2mg/ml PVA, plus 20ng/ml hVEGF-A) for an additional 72 hours. All differentiated cells were plated on VTN pre-coated culture plates. On day 5, endothelial cells stained with CD31 antibody (PECAM1, ThermoFisher Scientific, Wilmington, DE, USA) and CD144 antibody (CDH5, ThermoFisher Scientific) were analyzed by flow cytometry.

1.12 differentiation of hESCs into neurons

The neuron induction method is based on previous research³⁵. Briefly, hESCs were isolated by 1mg/ml collagenase IV treatment for 1 hour, resuspended in Embryoid Body (EB) medium (which is bFGF-free hESCs medium containing 2. mu.M of anti-morphine (dorsomorphin) and 2. mu.M of A-83-01b), and cultured in untreated polystyrene culture dishes for 7 days. Then, EBs were plated on Matrigel-coated 6-well culture plates, and the medium was changed to a Neural Progenitor Cell (NPC) medium, which was prepared by DMEM/F12: the neurobasal substance consists of 1:1, 1% N2, 1% B27, 1% NEAA, 1% GlutaMax, 2 μ g/ml heparin, and 2 μ M cyclopamine. The attached Ebs were cultured in NPC medium for 14 days, and the medium was changed every other day. Selecting by mechanical meansNeural progenitor cells were selected and then resuspended in NPC medium and the 6-well plates used were coated with ultra-low attachment surface (cat. No.3471, Corning, NY). For neuronal differentiation, suspended neural progenitor cells were treated with Accutase for 10 minutes and placed on poly-D-lysine coated coverslips in neuronal medium, which was a neural basal medium supplemented with 1% GlutaMax, 1% B27, 10ng/ml BDNF, and 10ng/ml GDNF. The medium was changed weekly and the next experiment was performed with neurons after 3 weeks. IF staining was used to assess the performance of TUJ1(801202, Biolegend) in nerve cells.

1.13 differentiation of hESCs into hepatocytes

The method complies with the method previously published in Nature Protocols³⁶. hESCs were isolated using 0.5mM EDTA (Life technologies, Camarillo, C, USA), seeded on VTN-N coated culture plates (Corning), and cultured in mTeSR1 medium for 48 hours. The medium was then refreshed daily for all subsequent steps. On days 1-2, the medium was replaced with fresh CDM-PVA (which was prepared from 0.5g PVA (Sigma), GlutaMAX (Invitrogen), 250ml IMDM (Invitrogen), 5ml chemically defined lipid concentrate (Invitrogen), 20. mu.l thioglycerol 97% (Sigma), 350. mu.l insulin (10 mg/ml; Roche), 250. mu.l transferrin (30 mg/ml; Roche) in 250. mu.l of transferrin plus activin A (100 ng/ml; R.sub.l/ml; R&D)、bFGF(80ng/ml；R&D)、BMP4(10ng/ml；R&D) And 10mM LY-294002(Promega) on day 3 cells were differentiated in RPMI medium supplemented with activin A (100ng/ml) and bFGF (80ng/ml) on days 4-6 cells were expanded in RPMI medium supplemented with activin A (50ng/ml) on day 7 cells were passaged and 105,000 cells per square centimeter were replated in RPMI containing activin A (50ng/ml) and Y-276322 HCl (10 μ M Selleck-chem, Houston, TX, USA and Munich, Germany), where the culture plates were coated with VTN-N, then on days 8-11 cells were cultured in RPMI + activin A (50ng/ml) into hepatocytes and analyzed after drug treatment using immunofluorescence staining, analysis of fetal proteins expressed in cells in Clakara α, Dakora, USA, Sankora protein expression in Santa, USA, Sankora protein, USA, and Biochemical analysis of liver cells。

1.14 immunofluorescence assay

Immunofluorescence assay was performed as previously described²⁹. Briefly, cells were washed with PBS, fixed in PBS with 4% formaldehyde solution for 10 minutes, and permeabilized with 0.3% Triton X-100 for 10 minutes. Then, the cells were stained with primary antibody at4 ℃ overnight. After washing with PBS, cells were washed with Alexa at room temperature

488 anti-rabbit IgG or anti-mouse IgG were cultured for 1 hour. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, 1. mu.g/ml).

1.15 Lentivirus (lentivirus) production and cell infection

293T cells at 10 per well⁶Individual cells were seeded in 6-well plates to produce lentiviruses. After 24 hours, 1. mu.g of pLKO _ AS3w.eGFP.bsd, 0.9. mu.g of pCMVR8.91, and 0.1. mu.g of pMD.G (National RNAi Core Facility, Taipei, Taiwa) were transfected into 293T cells using TurboFect transfection reagent (Thermo Fisher Scientific). At 24 hours post transfection, the medium was changed to virus harvest medium containing HG-DMEM, 10% FBS, and 1% BSA. Hbmmscs were plated in 6-well culture plates and then cultured with lentivirus (multiplicity of infection 10) for one day. On alternate days, cells were selected at 10. mu.g/ml blasticidin (Thermo Fisher Scientific). Then, the hBMMSCs overexpressing GFP were separated with a cell sorter (FACS ariaTM II, BD Biosciences) and cultured in a medium containing 10. mu.g/ml blasticidin for subsequent experiments.

1.16 transplantation of renal capsule

About 8x10⁵hESCs and 8x10⁵A mixture of hBMMSCs was injected under kidney capsules in 6-week old male NSG mice. After 5 weeks of transplantation, animals were sacrificed and tissues were removed, fixed with 10% formalin, paraffin embedded, H&E staining or IHC staining. The dyeing method is described in the materials and methods section. Anti-chicken GFP antibody (ab13970, Abcam, Cambridge, MA, USA) was used for hBMMSCs IHC staining for GFP overexpression.

1.17 Alamal blue (Alamar blue) test

The Alamar blue (Alamar blue) assay can determine relative cell number. In cells, the inventors added 1/10 of the Aromala blue survival test reagent to the total volume of the mixture (Biotium; Fremont, CA, USA). Cells were cultured overnight at 37 ℃. The resulting products were quantified by measuring the assay brightness (OD) at 570nm and 600nm, and the relative cell number was calculated.

1.18 statistical analysis

All statistical data are presented as mean ± standard deviation (s.d.) of at least three biological replicates. Statistically significant differences were assessed using either t-assays or single factor variance analysis, where p-values <0.05 were considered as having significant differences.

2. Results

2.1 Na in hESCs and hBMMSCs⁺/K⁺Differential representation of α subunits of ATPase

Since not all cancer signals overlap with hESC signals, the inventors determined that cardiac glycoside target gene Na⁺/K⁺Expression of ATPase to assess whether it can eliminate undifferentiated hESCs. Previous reports indicate that cardiac glycosides target Na⁺/K⁺-ATPase with anticancer effect^25,26. The inventors found, via western blot analysis, that hESCs more abundantly express Na than adult stem cells, such as human mesenchymal stem cells (hBMMSCs)⁺/K⁺-ATPase (fig. 1A). This finding suggests that hESCs may be more sensitive to cardiac glycosides than hBMMSCs because of their Na⁺/K⁺Differential presentation of ATPase.

2.2 Debrevifolin and hairy flower glycoside C induce hESCs cell apoptosis but not hBMMSCs

The inventors investigated whether cardiac glycosides affect viability of hESCs or other cell types. First, we treated undifferentiated hESCs with digitonin and ouabain C for 12 hours and 24 hours, respectively. Both digitonin (2.5 μ M) and floridin C (2.5 μ M) induced significant cell death in hESCs (fig. 1B). To investigate the cytotoxic effects of cardiac glycosides, the inventors determined the release of LDH in hESCs culture supernatants after 24 hours treatment with digitonin or digitonin C (fig. 1C). Both drugs significantly induced cytotoxic effects in hESCs (fig. 1C). Consistently, in another strain of hESCs cells, HUES6, both digitonin (2.5 μ M) and floridin C (2.5 μ M) induced cell death (fig. 6A) and cytotoxicity (fig. 6B).

In contrast, digitonin and ouabain C did not affect survival of hBMMSCs (fig. 1D). Both drugs had no cytotoxic effect on hBMMSCs as measured by the LDH cytotoxicity assay (fig. 1E).

Cardiac glycosides can be divided into two subgroups based on the natural structure of their lactone moieties^23,26. Digitonin and ouabain C belong to the cardiac glycosides (cardenolides) subgroup with butyl lactone²³. The inventor selects two medicines with pyranone ring from toad cardiac glycosides (bufadienolides) subgroup²³. The inventors treated hESCs with bufalin or proeleutherine a with hBMMSCs. The results were similar to those of digitoxin-and-hairy-glycoside-C treated cells, in which bufalin or prodelphinidin-A induced cytotoxicity in hESCs but not in hBMMSCs (FIG. 7).

To determine whether the cytotoxic effects of cardiac glycosides were selective for hESCs, the inventors also performed PI/annexin flow cytometry analysis. After 24 hours of treatment of the cells, the cell death rate after digitonin treatment reached 70%, while the cell death rate after ouabain C treatment reached 82% (fig. 2A). In contrast, more than 98% of the cells survived in digitonin or ouabain C treated hBMMSCs (fig. 2B). In addition, the inventors observed an increase in fragmentation patterns of PARP, apoptotic protease-3, and apoptotic protease-7 in digitonin-C and digitonin-C treated hESCs (FIG. 2C). In contrast, no disrupted apoptotic markers were detected in hBMMSCs treated with digitonin or ouabain C (fig. 2C). In addition to inducing cell death, the tumorigenic potential of the remaining hESCs is eliminated upon cell differentiation. Protein levels of Nanog were down-regulated after 12 hours of hESCs treated with digitonin or eriocitrin C (fig. 2D). Nanog is part of the core transcriptional regulatory network in ESCs. Deletion of Nanog in hESC induces extraembryonic lineage differentiation³⁷. It is reported that Nanog plays a key role in hESC omnipotence and self-renewal³⁸. These results indicate that cardiac glycosides induced cell death in hESCs, but not in hBMMSCs.

In addition, an Alamar blue (Alamar blue) test was performed. Digitonin, ouabain C, prodelphinogen a, digitoxin, and ouabain induced hESCs cell death in a dose-dependent manner (fig. 12).

2.3 differentiation of hBMMSCs into three lineages the differentiation ability was not affected by the treatment of digitonin or ouabain C

We demonstrate that cardiac glycosides do not affect the survival of hBMMSCs. MSCs are pluripotent cells and are expected to be used in regenerative medicine. MSCs can be specifically induced into osteoblasts, adipocytes and chondrocytes³⁹. To determine whether the differentiation potency of hBMMSCs was affected by digitonin or ouabain C, the inventors performed a differentiation assay. After 24 hours of treatment of hBMMSCs the digitonin or ouabain C was removed and the cells differentiated into three lineages. Notably, neither the digitonin nor the ouabain C affected the ability of hBMMSCs to differentiate into osteoblasts (fig. 3A), adipocytes (fig. 3B), and chondrocytes (fig. 3C). Based on these results, cardiac glycosides did not affect the pluripotency of hBMMSCs.

2.4 Digitalis Lanceolata or eriocitrin C did not induce cytotoxic effects in hESC-derived MSCs

To further validate the role of cardiac glycosides in hESCs and hESC-derived cell types, the inventors first selected hESC-derived MSCs (hESC-MSCs) as a model. Xu and colleagues provide a simple and rapid method to induce hESCs into hMSCs in a two-step process³⁰(FIG. 8A-FIG. 8C). The inventors differentiated H9 hESCs into MSCs and examined whether cardiac glycosides affected the viability of hESC-MSCs. hESC-MSCs were treated with digitonin and ouabain C for 12 hours and 24 hours, respectively. Neither digitonin (2.5 μ M) nor ouabain C (2.5 μ M) induced cell death in H9 hESC-MSCs. To investigate the cytotoxic effects of cardiac glycosides, the inventors determined that H9hESC-MSCs were treated with digitonin or eriocitrin C for 24 hoursLDH release in culture supernatant. Neither digitonin nor eriocitrin C affected the survival of H9hESC-MSCs (fig. 4B). In addition to digitonin and verbacoside C, the inventors also found that bufalin or proeleutherine a did not induce cytotoxicity (fig. 8D). This result is consistent with the effect observed in hBMMSCs (fig. 7).

To investigate whether cardiac glycosides induced cell death in H9hESC-MSCs, PI/annexin flow cytometry and western blot analysis were performed. Cell mortality was less than 2% after 24 hours treatment of hESC-MSCs with digitonin or ouabain C (fig. 4C). Consistently, no disrupted apoptotic markers were detected in hESC-MSCs treated with digitonin or ouabain C (fig. 4D). These results indicate that cardiac glycosides do not induce cell death in H9 hESC-MSCs. Further, Na⁺/K⁺ATPase is abundantly expressed in undifferentiated hESCs, but not in hESC-MSC (fig. 4E), which may confirm that cardiac glycoside toxicity is limited to undifferentiated hESCs.

2.5 Digitalis longifolacina glycoside and ouabain C did not or slightly induce cell death in hPSCs-derived endothelial cells, neurons, or hepatocyte endoderm

The inventors next tested whether digitonin and hairy flower glycoside C affect other hPSC differentiated cell types. CD34⁺/CD144⁺Chipsc-derived endothelial cell mesoderm lineages were gifted from dr, chiang and colleagues (national cheng Kung University, Tainan, Taiwan) (fig. 9A). Undifferentiated hiPSCs and hiPSCs-derived endothelial cells were exposed to digitonin or eriocitrin C for 24 hours. After treatment, digitonin and eriocitrin C induced cytotoxicity in hiPSCs (fig. 9B), but hiPSC-derived endothelial cells remained viable (fig. 9C). The inventors differentiated H9 hESCs into TUJ1 positive neurons, which belong to the ectoderm (fig. 9D), followed by treatment of these hESC neurons with digitonin or ouabain C for 24 hours. Digitonin and hairy flower glycoside C did not induce cytotoxicity in neuronal cells (FIG. 9E). Furthermore, the inventors differentiated hESCs into hepatocyte endoderm expressing AFP, which belongs to endoderm (fig. 9F). The results show that the drug may be mild, if any, in the hepatocyte endoderm cellsAny cell death was induced in the cells (less than 10%) (fig. 9G). Based on the above results, digitonin and ouabain C were likely to induce cell death specifically in undifferentiated hPSCs, but not in their differentiated progeny.

The inventors also tested the cytotoxic effects of other cardiac glycosides (including ouabain, digitoxin, proscilagin a, and bufalin) on undifferentiated pluripotent stem cells, hESCs and hPSCs, and various differentiated cells (including MSCs, neural cells, hepatocyte endoderm cells). The results are summarized below.

TABLE 1

2.6 Elephantopus scaber glycoside and ouabain C prevent teratoma formation in NSG mice

To investigate whether hESCs treated with cardiac glycosides lost the ability to form teratomas, hESCs treated with DMSO, digitoxin, or digitoxin C were transplanted alone into NSG mice for xenotransplantation (xenograft). The inventors found that the tumor weight was significantly reduced in the cardiac glycoside drug-treated group (fig. 5A and 5B). Thus, digitonin and ouabain C severely prevented most of the tumorigenicity in hESCs. Teratoma formation with hESCs treated with DMSO, digitonin, or digitonin C was shown to contain all three germ layers (fig. 5C and 10). These results confirm the versatility of these hESCs. The inventors have demonstrated that cardiac glycosides effectively prevent in vivo tumor formation.

Finally, to investigate whether the digitonin or ouabain C treated hBMMSCs remained in vivo, the inventors constructed hBMMSCs with GFP over-expression. hBMMSCs overexpressing GFP did not form tumors under the kidney capsule of NSG mice (fig. 11A). GFP-hBMMSCs remained at the site of transplantation, which was visualized by GFP IHC staining (FIG. 11A). The drug-treated mixture of hESCs and hBMMSCs was then injected under the kidney capsule of NSG mice. The tumor area was significantly inhibited in either digitonin or ouabain C treated group (fig. 11B), and the inventors still observed GFP-positive hBMMSCs (fig. 11C). These results demonstrate that the digitonin and ouabain C treated hBMMSCs remain under the kidney capsule in NSG mice.

3. Summary and discussion

In cell therapy, the remaining undifferentiated ESCs or iPSCs may raise safety concerns (teratomas) in their differentiated progeny for use of PSC-derived cells. Undifferentiated PSCs lose tumorigenicity upon terminal differentiation. However, the remaining undifferentiated PSCs must be removed prior to application to ESCs and iPSCs cell therapy⁴⁰. In this study, the inventors' data demonstrated the cytotoxic effects of cardiac glycosides in hESCs (fig. 1B, 1C, 2A, 2C, 6, 12). This phenomenon was not observed in hBMMSCs (fig. 1D, 1E, 2B, 2C). Most importantly, these drugs did not affect the differentiation capacity of stem cells (fig. 3). Similar effects of cardiac glycosides are shown in hESC-derived progeny. The viability of hESC-MSCs, hESC-neurons, and hiPSC-endothelial cells was also not affected by digitonin and ouabain C (fig. 4, fig. 9). In addition, in vivo experiments confirmed that digitonin and ouabain C were effective in preventing teratoma formation (fig. 5). The inventors' work describes for the first time the cytotoxic effect and tumor prevention ability of cardiac glycosides in hESCs. Digitonin and hairy flower glycoside C are also the first FDA-approved drugs with cytotoxicity in hESCs.

To overcome the risk of teratoma formation in regenerative medicine, several strategies have been proposed^41,42. Antibody sorting and cytotoxic antibody strategies can be simple, but are limited in efficiency due to single cell isolation requirements or antibody batch variation⁷ _, ^17,18,43. These methods are also costly. Another strategy is based on the manipulation of genes that track target cells^10,12,44,45However, these methods are laborious and expensive. Most importantly, insertional mutagenesis is a biosafety threat to these genetically altered cells in clinical use⁴². Chemical elimination (chemical elimination) strategies are fast, robust, and efficient, and are also the most cost-effective methods. The chemical method does not require cell sorting and any genetic manipulation.

In addition to small molecule methods, one study also used metabolic selection(Metabolic selection) enrichment of PSC-derived cardiomyocytes¹³. The authors provide an interesting method to purify cardiomyocytes from PSCs in glucose-depleted and lactate-enriched culture conditions. This method is well suited for producing high purity cardiomyocytes. However, other cell types may require more tests to determine the specific metabolic utility of the cell type. This strategy is attractive, but only applicable to a very small number of cell types. The inventors eliminated undifferentiated hESCs with CG drugs, and these drugs did not affect the viability of several different cell types (i.e., MSCs, endothelial cells, neurons). Since CGs are easy to purchase and cost effective, this approach is quite convenient for future applications. Digitonin and hairy flower glycoside C are potent small molecules that inhibit tumorigenic hESCs in culture, the results of which are shown in teratoma formation experiments (fig. 5). Na (Na)⁺/K⁺Expression of the ATPase subunit varies between cancer and normal cells or tissues²³. Furthermore, the inventors' data indicate that Na⁺/K⁺The expression of-ATPase subunits also varied between undifferentiated and differentiated cells (fig. 1A, fig. 4E). In this study, the inventors have discovered novel applications of cardiac glycosides that could improve the major concerns of hPSCs cell therapy by preventing teratoma formation.

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Claims

1. A method of removing undifferentiated pluripotent stem cells from a sample containing such cells, the method comprising exposing the sample to an effective amount of a cardiac glycoside.

2. The method of claim 1, wherein the cardiac glycoside is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

wherein L is a lactone group;

R₁、R₄and R₅Independently is H or OH;

R₂is CH₃Or CH₂OH；

R₄is H or OH;

R₅is H or OH;

R₆is CH₃(ii) a And

3. The method of claim 2, wherein the lactone group is an unsaturated, five or six membered lactone ring.

4. The method of claim 2, wherein the sugar residue is selected from the group consisting of rhamnose, glucose, digitoxin, digitose, gluco-oligosaccharide (digginose), rhabdomyo-pyranose (sarmentose), valarose (vallarose), and fructose.

5. The method of claim 2, wherein the sugar residue is unsubstituted' or substituted with an acyl group, an amino group, a halo group, and/or an amido group.

6. The method of claim 2, wherein X is a glycoside of two digitoxins, one acetyl-digitoxin, and one glucose; or X is a glycoside of three digitoxoses; or X is a rhamnose glycoside.

7. The method of claim 2, wherein the compound is selected from the group consisting of:

(I) -1 (digitoxin);

(I) -2 (hairy flower glycoside C);

(I) -3 (ouabain);

(I) -4 (digitoxin);

(I) -5 (digitoxin);

(I) -6 (bufalin); and

(I) -7 (proeleutherogen A).

8. The method of any one of claims 1-7, wherein the undifferentiated pluripotent stem cells are selected from the group consisting of Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs).

9. The method of any one of claims 1-8, wherein the undifferentiated pluripotent stem cells exhibit a cellular marker selected from the group consisting of Na⁺/K⁺-ATPase, Nanog, Oct4, Sox2, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and combinations thereof.

10. The method of any one of claims 1-9, wherein the sample further comprises differentiated cells.

11. The method of claim 10, wherein the method further comprises culturing the differentiated cells to provide a population of the differentiated cells.

12. The method of claim 10, wherein the differentiated cells are Mesenchymal Stem Cells (MSCs).

13. The method of claim 12, wherein the differentiated cells exhibit a cellular marker selected from the group consisting of CD44⁺、CD73⁺、CD90⁺、CD105⁺And combinations thereof.

14. The method of claim 13, wherein the differentiated cell is CD45^-、CD34^-、CD11b^-、CD19^-And/or HLA-DR^-。

15. The method of claim 10, wherein the differentiated cell is selected from the group consisting of osteoblasts, adipocytes, chondrocytes, endothelial cells, neural cells, and hepatocytes.

16. A method of making a differentiated cell, comprising:

(iii) the remaining differentiated cells may be cultured as necessary.

17. The method of claim 16, wherein the cardiac glycoside is a compound of formula (I) as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

18. The method of claim 16 or 17, wherein the undifferentiated pluripotent stem cells are selected from the group consisting of Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs).

19. The method of claim 16 or 17, wherein the differentiated cells are Mesenchymal Stem Cells (MSCs).

20. The method of claim 19, wherein the differentiated cells exhibit a cellular marker selected from the group consisting of CD44⁺、CD73⁺、CD90⁺、CD105⁺And combinations thereof.

21. The method of claim 20, wherein the differentiated cell is CD45^-、CD34^-、CD11b^-、CD19^-And/or HLA-DR^-。

22. The method of claim 16 or 17, wherein the differentiated cell is selected from the group consisting of osteoblasts, adipocytes, chondrocytes, endothelial cells, neural cells, and hepatocytes.

23. A method for treating a subject in need of cell therapy, comprising administering to the subject a population of cells comprising differentiated cells, wherein the population of cells is exposed to a cardiac glycoside prior to administration to the subject, thereby removing, if any, undifferentiated cells present in the population of cells.

24. The method of claim 23, wherein the cardiac glycoside is a compound of formula (I) as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

25. The method of claim 23 or 24, wherein the undifferentiated pluripotent stem cells are selected from the group consisting of Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs).

26. The method of claim 23 or 24, wherein the differentiated cells are Mesenchymal Stem Cells (MSCs).

27. The method of claim 26, wherein the differentiated cells exhibit a cell marker selected from the group consisting of CD44⁺、CD73⁺、CD90⁺、CD105⁺And combinations thereof.

28. The method of claim 27, wherein the differentiated cell is CD45^-、CD34^-、CD11b^-、CD19^-And/or HLA-DR^-。

29. The method of claim 23, wherein the differentiated cell is selected from the group consisting of an osteoblast, an adipocyte, a chondrocyte, an endothelial cell, a neural cell, and a hepatocyte.

30. Use of a cardiac glycoside for the manufacture of a composition for inducing cell death of undifferentiated pluripotent stem cells.

31. The use of claim 30, wherein the cardiac glycoside is a compound of formula (I) as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

32. The method of claim 30 or 31, wherein the undifferentiated pluripotent stem cells are selected from the group consisting of Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs).

33. A composition for use in cell therapy comprising a cardiac glycoside and a population of cells comprising differentiated cells.

34. The composition of claim 33, wherein the cardiac glycoside is a compound of formula (I) as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

35. The composition of claim 33 or 34, wherein the population of cells is exposed to a cardiac glycoside prior to administration to a subject in need thereof, thereby substantially removing, if any, undifferentiated cells present in the population of cells.

36. A method for treating a teratoma in a subject in need thereof, comprising administering to the subject an effective amount of a cardiac glycoside.

37. The method of claim 36, wherein the cardiac glycoside is a compound of formula (I) as defined in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.